E. coli plasmids have long been an important source of recombinant DNA molecules used by researchers and by industry. Therapeutic plasmids and plasmid based bacterial expression vectors typically contain a pMB1, ColE1 or pBR322 derived replication origin. In general, the replication origin needs to be protected from read-through transcription from adjacent genes, for example from cryptic promoters in an antigen gene insert, to prevent plasmid destabilization or reduced copy number (Engels P, Meyer P. 1993. Biotechniques 14: 324-5). This may be accomplished by inclusion of shielding transcriptional terminators. For example, transcriptional terminators shielding the prokaryotic replication origin from insert mediated transcriptional read-through reduce insert toxicity due to eukaryotic gene transcription (Brosius J. 1984. Gene, 27: 161-72; Chen W, Kallio P T, Bailey J E. 1993. Gene 130: 15-22; Chen J D, Morrision D A. 1987. Gene 55: 179-87).
However, a gene insert may contain DNA structures, proteins or peptides that are toxic to the host cell. Hydrophobic membrane spanning peptides are particularly toxic to bacteria. Expression of these peptides, by gene insert containing cryptic bacterial promoters or vector encoded cryptic bacterial promoters, will not be prevented by transcriptional terminators shielding the prokaryotic replication origin.
Translation of an insert encoded sense strand toxic peptide may be prevented by introduction of a reverse oriented promoter after the insert, to generate translation-disrupting antisense RNA (Weiner D B, Zhang D, Cohen A. 2005. U.S. Pat. No. 6,881,558; reviewed by Saida F, Uzan M, Odaert B, Bontems F. 2006. Current Protein Peptide Science 7: 47-56). This has the disadvantage of requiring vector modification to insert the promoter. As well, since not all insert borne toxic peptides are expressed from the sense strand, this strategy will detrimentally increase expression of toxic peptides encoded by the antisense strand.
Alternatively, stabilization of a plasmid containing an insert encoded toxic peptide may be achieved through insertion of an intron to disrupt bacterial production of a known toxic protein (Boyd A C, Popp F, Michaelis U, Davidson H, Davidson-Smith H, Doherty A, McLachlan G, Porteous D J, Seeber S. 1999. J. Gene Med. 1: 312-21). This method has the disadvantage of requiring vector modification to insert the intron, and requires knowledge of both the exact location of the toxic peptide, as well as a site within this toxic peptide in which insertion of an intron will disrupt toxicity.
Alternatively, stabilization of a plasmid containing an insert encoded toxic peptide may be achieved through expression of antisense RNA from a second plasmid. The antisense RNA from the second plasmid will bind to the mRNA encoding the toxic peptide (Futterer J, Gordon K, Pfeiffer P, Hohn T. 1988. Gene 67: 141-5). A disadvantage of this strategy is required dual selection for two plasmids, and two plasmids containing identical inserts would likely recombine (Weiner et al., Supra, 2005). As well, for plasmid production, contamination of the target plasmid with the antisense-RNA encoding second replicon would be unacceptable. The increased metabolic burden associated with maintaining a second plasmid is also undesired.
Even in view of the prior art there is a need to reduce insert-associated toxicity, to prevent plasmid destabilization or reduced copy number that would affect plasmid production, or plasmid directed protein production.